Flanking Bases Research Articles

Two coexisting pseudo-mirror heteromolecular telomeric G-quadruplexes in opposite loop progressions differentially recognized by a low equivalent of Thioflavin T.

The final 3′-terminal residue of the telomeric DNA G-overhang is inherently less precise. Here, we describe how alteration of the last 3′-terminal base affects the mutual recognition between two different G-rich oligomers of human telomeric DNA in the formation of heteromolecular G-quadruplexes (hetero-GQs). Associations between three- and single-repeat fragments of human telomeric DNA, target d(GGGTTAGGGTTAGGG) and probe d(TAGGGT), in Na+ solution yield two coexisting forms of (3 + 1) hybrid hetero-GQs: the kinetically favourable LLP-form (left loop progression) and the thermodynamically controlled RLP-form (right loop progression). However, only the adoption of a single LLP-form has been previously reported between the same probe d(TAGGGT) and a target variant d(GGGTTAGGGTTAGGGT) having one extra 3′-end thymine. Moreover, the flanking base alterations of short G-rich probe variants also significantly affect the loop progressions of hetero-GQs. Although seemingly two pseudo-mirror counter partners, the RLP-form exhibits a preference over the LLP-form to be recognized by a low equivalent of fluorescence dye thioflavin T (ThT). To a greater extent, ThT preferentially binds to RLP hetero-GQ than with the corresponding telomeric DNA duplex context or several other representative unimolecular GQs.

Sequence Dependent Repair of 1,N6-Ethenoadenine by DNA Repair Enzymes ALKBH2, ALKBH3, and AlkB.

Mutation patterns of DNA adducts, such as mutational spectra and signatures, are useful tools for diagnostic and prognostic purposes. Mutational spectra of carcinogens derive from three sources: adduct formation, replication bypass, and repair. Here, we consider the repair aspect of 1,N6-ethenoadenine (εA) by the 2-oxoglutarate/Fe(II)-dependent AlkB family enzymes. Specifically, we investigated εA repair across 16 possible sequence contexts (5′/3′ flanking base to εA varied as G/A/T/C). The results revealed that repair efficiency is altered according to sequence, enzyme, and strand context (ss- versus ds-DNA). The methods can be used to study other aspects of mutational spectra or other pathways of repair.

Fluorene‐Labeled 2'‐Deoxyuridine as an Environmentally Sensitive Probe for Detection of an Abasic Site

AbstractThe fluorescence properties of the fluorene‐labeled 2'‐deoxyuridine derivative UF are sensitive to the polarity and viscosity of its solvent. We incorporated this environment‐sensitive unit into the central position of oligodeoxynucleotides (ODNs) and placed various flanking bases (FBs) around the UF residue, then investigated the changes in fluorescence that occurred when these ODN probes encountered DNA targets containing an abasic site. The ODN probe featuring cytosine‐FBs turned on its fluorescence when contacting the fully matched target DNA sequence, but turned off its fluorescence dramatically when the target DNA contained an abasic site.

Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms

Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases, DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substrate-recognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.

Switch-on effect on conformation-specific arylamine-DNA adduct by cyclometalated Ir(III) complexes.

Arylamines are known to form covalent-DNA adducts upon metabolic activation. These covalent adducts adopt different conformational attributes, viz., major groove (B), stacked (S), and minor groove (W), and lead to different types of mutations. The conformation depends on the flanking and next flanking bases at the 3' position of the adduct. Early detection of these conformations by simple probes is an ideal and challenging task. Here, we have reported two Ir(III)-based cyclometalated complexes, viz., [Ir(ppy)2(imiphen)]+ (1) (ppy: 2-phenylpyridine; imiphen: 2-(1H-imidazol-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(ppy)2(furphen)]+ (2) (furphen: 2-(furan-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its interaction with N-acetyl-2-aminofluorene-dG (AAF-dG). The sequences used in this work are NarI sequence (-CG1G2CG3CX-) in which Gs are modified with AAF and X is either C or T. Luminescence studies reveal that the Ir(III) complexes bind to AAF-dG adduct with high specificity toward G1 and G3 compared to G2 and unmodified control. The selectivity also depends on the next flanking base as cytosine favors G3AAF, while thymine favors G1AAF in complex 1 and vice versa for complex 2. The quenching studies confirm that Ir(III) complexes bind with AAF-dG sequences through the minor groove. The outcome of this work reveals that the switch-on effect by the complexes can be utilized for determining the conformational heterogeneity of the adduct and also for similar covalent-DNA adducts.

Selective Recognition of Conformation-Specific Arylamine-DNA Adduct in Frameshift Model by [Ru(phen)2(dppz)]2.

Arylamine modification of guanine base at the G3 position in the NarI sequence (-G1G2CG3CC-) causes a frameshift mutation. Polymerase and 19F NMR studies have shown that the next flanking base at the 3' position to the G3 adduct modulates the mutational outcome because of its different conformations. Here, we have studied the interaction of the 16-mer NarI sequence (5'-CTCTCG1G2CG3CXATCAC-3') (G3 = N-acetyl-2-aminofluorene (AAF)-dG and X is either C or T) with [Ru(phen)2(dppz)]2+ (phen = 1,10-phenanthroline and dppz = dipyrido[3,2-a:2',3'-c]phenazine). Interaction studies between isomers of Ru(II) and two oligonucleotide models, viz., (a) full duplex, and (b) slipped mutagenic intermediate (SMI), have been carried out. Luminescence studies reveal that the sensitivity of Ru(II) with an adduct increases 2- to 3-fold compared to that of control in full duplex. In SMI, the sensitivity of Ru(II) varies with the next flanking base and in the order of -GAAFCC > -GAAFCT. Microscale thermophoretic data reveal that in full duplex Λ-Ru binds to -GAAFCT- by 13- and 4-fold stronger than its control and -GAAFCC-, respectively. In SMI, Δ-Ru binds to -GAAFCC- (41% stacked (S) conformer) by 3-fold while -GAAFCT- (86% major groove (B) conformer) weakens the binding of Λ-Ru by 250-fold compared to the control. The results presented here reveal that the binding of Ru(II) not only depends on conformations of the AAF-dG adduct but also is isomer-centric and might be helpful in determining the conformational heterogeneity of other covalent aryl/heterocyclic amine-DNA adducts.

Corrigendum: Photochemical Formation of Cyclobutane Pyrimidine Dimers in DNA through Electron Transfer from a Flanking Base.

Electron transfer (ET) to a pyrimidine base from external moieties is a common step involved in the quenching or repair of the cyclobutane pyrimidine dimer (CPD). In contrast, we present a pathway that is initiated by an ET from a flanking guanine base to a pyrimidine base, leading to the formation of a CPD. We studied a T5mCG sequence with a methylated cytosine and our results demonstrate that the pathway involves the formation of an exciplex and intersystem crossings. This pathway also provides an explanation for why the mutational hot spots are correlated with the methylated CpG sequences, which has been a significant issue in cancer research.

Structural insights into the mechanism of double strand break formation by Hermes, a hAT family eukaryotic DNA transposase.

Some DNA transposons relocate from one genomic location to another using a mechanism that involves generating double-strand breaks at their transposon ends by forming hairpins on flanking DNA. The same double-strand break mode is employed by the V(D)J recombinase at signal-end/coding-end junctions during the generation of antibody diversity. How flanking hairpins are formed during DNA transposition has remained elusive. Here, we describe several co-crystal structures of the Hermes transposase bound to DNA that mimics the reaction step immediately prior to hairpin formation. Our results reveal a large DNA conformational change between the initial cleavage step and subsequent hairpin formation that changes which strand is acted upon by a single active site. We observed that two factors affect the conformational change: the complement of divalent metal ions bound by the catalytically essential DDE residues, and the identity of the –2 flanking base pair. Our data also provides a mechanistic link between the efficiency of hairpin formation (an A:T basepair is favored at the –2 position) and Hermes' strong target site preference. Furthermore, we have established that the histidine residue within a conserved C/DxxH motif present in many transposase families interacts directly with the scissile phosphate, suggesting a crucial role in catalysis.

Cover Feature: Photochemical Formation of Cyclobutane Pyrimidine Dimers in DNA through Electron Transfer from a Flanking Base (ChemPhysChem 13/2018)

Cover Feature: Photochemical Formation of Cyclobutane Pyrimidine Dimers in DNA through Electron Transfer from a Flanking Base (ChemPhysChem 13/2018)

Photochemical Formation of Cyclobutane Pyrimidine Dimers in DNA through Electron Transfer from a Flanking Base.

Electron transfer (ET) to a pyrimidine base from external moieties is a common step involved in the quenching or repair of the cyclobutane pyrimidine dimer (CPD). In contrast, we present a pathway that is initiated by an ET from a flanking guanine base to a pyrimidine base, leading to the formation of a CPD. We studied a T5mCG sequence with a methylated cytosine and our results demonstrate that the pathway involves the formation of an exciplex and intersystem crossings. This pathway also provides an explanation for why the mutational hot spots are correlated with the methylated CpG sequences, which has been a significant issue in cancer research.

Conformational Preference and Fluorescence Response of a C-Linked C8-Biphenyl-Guanine Lesion in the NarI Mutational Hotspot: Evidence for Enhanced Syn Adduct Formation.

Aromatic chemical carcinogens can undergo enzymatic transformations to produce a range of electrophilic species that attach covalently to the C8-site of 2'-deoxyguanosine (dG) to afford C8-dG adducts. The most studied C8-dG adducts are formed from arylamines and contain a N-linkage separating the dG from the C8-aryl moiety. Other carcinogenic species result in direct aryl ring attachment to the dG moiety, resulting in C-linked adducts. The resulting C-linked adducts have reduced conformational flexibility compared to the corresponding N-linked C8-dG adducts, which can alter their orientation in the DNA duplex. Described herein are structural studies of a fluorescent C-linked 4-fluorobiphenyl-dG (FBP-dG) that has been incorporated into the reiterated G3-postion of the 12-mer NarI sequence and those containing other 5'-flanking nucleobases. FBP-dG displays a strong preference for adopting a syn conformation in the fully paired NarI duplex to produce an intercalated structure that exhibits stacking interactions between the C-linked biphenyl and the flanking bases. FBP-dG is also shown to significantly stabilize the slippage mutagenic intermediate (SMI) duplex containing the lesion and 5'-flanking base within a 2-base bulge. FBP-dG exhibits fluorescence sensitivity to SMI duplex formation that can readily distinguish it from the fully paired duplex. Molecular dynamics simulations and optical spectroscopy for the NarI oligonucleotides containing the C-linked FBP-dG predict increased rigidity of the biphenyl in the syn conformation. The greater propensity to generate the promutagenic syn conformation for the C-linked FBP-dG adduct compared to the N-linked 4-aminobiphenyl-dG adduct (ABP-dG) suggests greater mutagenicity for the C-linked analogue. These results highlight the effect of the adduct linkage type on the conformational properties of adducted DNA. The turn-on emission response of FBP-dG in the SMI duplex may be a powerful tool for monitoring SMI formation in the NarI sequence upon synthesis with DNA polymerases.

Sensitive and specific detection of microRNAs based on two-stage amplification reaction using molecular beacons as turn-on probes

In this study, a rapid, sensitive, and specific assay for detecting miRNAs was developed based on a two-stage amplification reaction (TSAR) using molecular beacons (MBs) as turn-on probes. In the TSAR, different miRNAs can be converted to the same reporter oligonucleotides (Y), which can hybridize with the same MB. Therefore, in combination with specific templates, this method can be applied to multiplex miRNA detection by simply using the same MB. The loop region of the MB was screened by computer simulation methods. In particular, to improve the specificity of the MB in real sample analysis, the maximum similarity of the MB loop region to the human genome and human transcriptome is less than 70%. Two MBs were designed in this study. MB I, with nine flanking base pairs in its stem region, was used for real-time monitoring of the production of Y during the TSAR. MB II, with five flanking base pairs in its stem region, was used to detect the production of Y at the end of the TSAR. This assay exhibited high sensitivity with a limit of detection of 2.0 × 10−16M and 6.7 × 10−16M using MB I and MB II as turn-on probes, respectively. In addition, this assay can clearly discriminate single base differences in miRNA sequences, and the TSAR can be completed under isothermal conditions. Accordingly, the isothermal reaction conditions and simple fluorescence measurement can greatly contribute to the development of a fast point-of-care detection system.

The Role of +4U as an Extended Translation Termination Signal in Bacteria.

Termination efficiency of stop codons depends on the first 3′ flanking (+4) base in bacteria and eukaryotes. In both Escherichia coli and Saccharomyces cerevisiae, termination read-through is reduced in the presence of +4U; however, the molecular mechanism underlying +4U function is poorly understood. Here, we perform comparative genomics analysis on 25 bacterial species (covering Actinobacteria, Bacteriodetes, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Proteobacteria, and Spirochaetae) with bioinformatics approaches to examine the influence of +4U in bacterial translation termination by contrasting highly- and lowly-expressed genes (HEGs and LEGs, respectively). We estimated gene expression using the recently formulated Index of Translation Elongation, ITE, and identified stop codon near-cognate transfer RNAs (tRNAs) from well-annotated genomes. We show that +4U was consistently overrepresented in UAA-ending HEGs relative to LEGs. The result is consistent with the interpretation that +4U enhances termination mainly for UAA. Usage of +4U decreases in GC-rich species where most stop codons are UGA and UAG, with few UAA-ending genes, which is expected if UAA usage in HEGs drives up +4U usage. In HEGs, +4U usage increases significantly with abundance of UAA nc_tRNAs (near-cognate tRNAs that decode codons differing from UAA by a single nucleotide), particularly those with a mismatch at the first stop codon site. UAA is always the preferred stop codon in HEGs, and our results suggest that UAAU is the most efficient translation termination signal in bacteria.

Radioresistance of GGG sequences to prompt strand break formation from direct-type radiation damage.

As humans, we are constantly exposed to ionizing radiation from natural, man-made and cosmic sources which can damage DNA, leading to deleterious effects including cancer incidence. In this work, we introduce a method to monitor strand breaks resulting from damage due to the direct effect of ionizing radiation and provide evidence for sequence-dependent effects leading to strand breaks. To analyze only DNA strand breaks caused by radiation damage due to the direct effect of ionizing radiation, we combined an established technique to generate dehydrated DNA samples with a technique to analyze single-strand breaks on short oligonucleotide sequences via denaturing gel electrophoresis. We find that direct damage primarily results in a reduced number of strand breaks in guanine triplet regions (GGG) when compared to isolated guanine (G) bases with identical flanking base context. In addition, we observe strand break behavior possibly indicative of protection of guanine bases when flanked by pyrimidines and sensitization of guanine to strand break when flanked by adenine (A) bases in both isolated G and GGG cases. These observations provide insight into the strand break behavior in GGG regions damaged via the direct effect of ionizing radiation. In addition, this could be indicative of DNA sequences that are naturally more susceptible to strand break due to the direct effect of ionizing radiation.

Conformational Insights into the Mechanism of Acetylaminofluorene-dG-Induced Frameshift Mutations in the NarI Mutational Hotspot.

Frameshift mutagenesis encompasses the gain or loss of DNA base pairs, resulting in altered genetic outcomes. The NarI restriction site sequence 5'-G1G2CG3CX-3' in Escherichia coli is a well-known mutational hotspot, in which lesioning of acetylaminofluorene (AAF) at G3* induces a greater -2 deletion frequency than that at other guanine sites. Its mutational efficiency is modulated by the nature of the nucleotide in the X position (C ∼ A > G ≫ T). Here, we conducted a series of polymerase-free solution experiments that examine the conformational and thermodynamic basis underlying the propensity of adducted G3 to form a slipped mutagenic intermediate (SMI) and its sequence dependence during translesion synthesis (TLS). Instability of the AAF-dG3:dC pair at the replication fork promoted slippage to form a G*C bulge-out SMI structure, consisting of S- ("lesion stacked") and B-SMI ("lesion exposed") conformations, with conformational rigidity increasing as a function of primer elongation. We found greater stability of the S- compared to the B-SMI conformer throughout TLS. The dependence of their population ratios was determined by the 3'-next flanking base X at fully elongated bulge structures, with 59% B/41% S and 86% B/14% S for the dC and dT series, respectively. These results indicate the importance of direct interactions of the hydrophobic AAF lesion with the 3'-next flanking base pair and its stacking fit within the -2 bulge structure. A detailed conformational understanding of the SMI structures and their sequence dependence may provide a useful model for DNA polymerase complexes.

Structural Chemistry of Human RNA Methyltransferases.

RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

The effect of sequence context on the activity of cytosine DNA glycosylases.

We have prepared single (N204D) and double (N204D:L272A) mutants of human uracil DNA glycosylase (hUDG), generating two cytosine DNA glycosylases (hCDG and hCYDG). Both these enzymes are able to excise cytosine (but not 5-methylcytosine), when this base is part of a mismatched base pair. hCDG is more active than the equivalent E. coli enzyme (eCYDG) and also has some activity when the cytosine is paired with guanine, unlike eCYDG. hCDG also has some activity against single stranded DNA, while having poor activity towards an unnatural base pair that forces the cytosine into an extrahelical conformation (in contrast to eCYDG for which a bulky base enhances the enzyme's activity). We also examined how sequence context affects the activity of these enzymes, determining the effect of flanking base pairs on cleavage efficiency. An abasic site or a hexaethylene glycol linker placed opposite the target cytosine, also causes an increase in activity compared with an AC mismatch. Flanking an AC mismatch with GC base pairs resulted in a 100-fold decrease in excision activity relative to flanking AT base pairs and the 5'-flanking base pair had a greater effect on the rate of cleavage. However, this effect is not simply due to the stability of the flanking base pairs as adjacent GT mismatches also produce low cleavage efficiency.

Conformational insights into the lesion and sequence effects for arylamine-induced translesion DNA synthesis: 19F NMR, surface plasmon resonance, and primer kinetic studies.

Adduct-induced DNA damage can affect transcription efficiency and DNA replication and repair. We previously investigated the effects of the 3′-next flanking base (G*CT vs G*CA; G*, FABP, N-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl; FAF, N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) on the conformation of arylamine-DNA lesions in relation to E. coli nucleotide excision repair (JainV., HiltonB., LinB., PatnaikS., LiangF., DarianE., ZouY., MackerellA. D.Jr., and ChoB. P. (2013) Nucleic Acids Res., 41, 869−88023180767). Here, we report the differential effects of the same pair of sequences on DNA replication in vitro by the polymerases exofree Klenow fragment (Kf-exo–) and Dpo4. We obtained dynamic 19F NMR spectra for two 19-mer modified templates during primer elongation: G*CA [d(5′-CTTACCATCG*CAACCATTC-3′)] and G*CT [d(5′-CTTACCATCG*CTACCATTC-3′)]. We found that lesion stacking is favored in the G*CT sequence compared to the G*CA counterpart. Surface plasmon resonance binding results showed consistently weaker affinities for the modified DNA with the binding strength in the order of FABP > FAF and G*CA > G*CT. Primer extension was stalled at (n) and near (n – 1 and n + 1) the lesion site, and the extent of blockage and the extension rates across the lesion were influenced by not only the DNA sequences but also the nature of the adduct’s chemical structure (FAF vs FABP) and the polymerase employed (Kf-exo– vs Dpo4). Steady-state kinetics analysis with Kf-exo– revealed the most dramatic sequence and lesion effects at the lesion (n) and postinsertion (n + 1) sites, respectively. Taken together, these results provide insights into the important role of lesion-induced conformational heterogeneity in modulating translesion DNA synthesis.

Structural and Thermodynamic Insight into Escherichia coli UvrABC-Mediated Incision of Cluster Diacetylaminofluorene Adducts on the NarI Sequence

Cluster DNA damage refers to two or more lesions in a single turn of the DNA helix. Such clustering may occur with bulky DNA lesions, which may be responsible for their sequence-dependent repair and mutational outcomes. Here we prepared three 16-mer cluster duplexes in which two fluoroacetylaminofluorene adducts (dG-FAAF) are separated by zero, one, and two nucleotides in the Escherichia coli NarI mutational hot spot (5'-CTCTCG1G2CG3CCATCAC-3'): 5'-CG1*G2*CG3CC-3', 5'-CG1G2*CG3*CC-3', and 5'-CG1*G2CG3*CC-3' (G* = dG-FAAF), respectively. We conducted spectroscopic, thermodynamic, and molecular dynamics studies of these di-FAAF duplexes, and the results were compared with those of the corresponding mono-FAAF adducts in the same NarI sequence [Jain, V., et al. (2012) Nucleic Acids Res. 40, 3939-3951]. Our nucleotide excision repair results showed the diadducts were more reparable than the corresponding monoadducts. Moreover, we observed dramatic flanking base sequence effects on their repair efficiency in the following order: NarI-G2G3 > NarI-G1G3 > NarI-G1G2. The nuclear magnetic resonance, circular dichroism, ultraviolet melting, and molecular dynamics simulation results revealed that in contrast to the monoadducts, diadducts produced a synergistic effect on duplex destabilization. In addition, dG-FAAF at G2G3 and G1G3 destacks the neighboring bases, with greater destabilization occurring with the former. Overall, the results indicate the importance of base stacking and related thermal and thermodynamic destabilization in the repair of bulky cluster arylamine DNA adducts.

Highly Efficient Quencher‐Free Molecular Beacon Systems Containing 2‐Ethynyldibenzofuran‐ and 2‐Ethynyldibenzothiophene‐Labeled 2′‐Deoxyuridine Units

We have prepared two fluorescent DNA probes--UDBF and UDBT, containing 2-ethynyldibenzofuran and 2-ethynyldibenzothiophene moieties, respectively, covalently attached to the base dU--and incorporated them in the central positions of oligodeoxynucleotides (ODNs) so as to develop new types of quencher-free linear beacon probes and investigate the effect of functionalization of the fluorene scaffold on the photophysical properties of the fluorescent ODNs. The ODNs containing adenine flanking bases (FBs) displayed a selective fluorescence "turn-off" response to mismatched targets with guanine bases; this suggests that these probes could be used as base-discriminating fluorescent nucleotides. On the other hand, we observed a "turn-on" response to matched targets when the UDBF and UDBT units of ODNs containing pyrimidine-based FBs were positioned opposite the four natural nucleobases. In particular, an ODN incorporating UDBT and cytosine FBs has potential use in single-nucleotide polymorphism typing.